FUNCTIONAL DETERMINANTS OF G PROTEIN-COUPLED RECEPTORS PROJECT SUMMARY/ABSTRACT The long-term goal of this work is to rationally manipulate G protein-coupled receptor (GPCRs) activation with the hope to develop novel therapeutic approaches in this major family of transmembrane receptors and drug targets. Our hypothesis is that evolutionary divergence patterns can reveal the roles that GPCR sequence positions play, individually or together, in order to mediate ligand binding, allosteric conformational switching, and finally ligand-biased activation of efferent signaling pathways, which can be G protein-dependent or independent. In the past funding period, computational analysis of such evolutionary patterns revealed: Intramolecular allosteric communication in the transmembrane domain of dopamine D2R receptor; Modular components of an allosteric switch controlling B2AR functional selectivity; A new non-canonical cAMP- independent signaling pathway in that same receptor; and Ligand specificity determinants in the extracellular domain of metabotropic glutamate receptors (MGluR). Technical progress led to: increased accuracy to assess the impact of coding mutations in proteins through a first-principle equation for the evolutionary variations of genotype and phenotype; The generalization of our analyses of evolutionary divergence to consider co-varying residues; and new methods to unravel complex simultaneous assay readouts to stratify drug effects on GPCRs. Together these and other data support new aims that combine biological and algorithmic goals: 1. To titrate mutationally the signaling bias of bioamine receptors. 2. To uncover allosteric mediators in metabotropic glutamate receptors. 3. To identify a systematic role of GPCR mutations in cancer. The outcome should reveal new aspects of the molecular basis of signaling in an important family of pharmaceutical targets. It will also link sequence and structure genomics databases to the molecular basis of function and to the rational re- design of protein function?key steps towards manipulating cellular pathways.